The full nomenclature of the subject matter of the present invention involves lengthy terms. It is customary for those skilled in the art to abbreviate oligoadenylate analogues and related terms in a manner well-known to the art. These general and customary abbreviations are set forth herein below and may be utilized in the text of this specification.
Abbreviations
2-5A, 2',5'-oligoadenylate or p.sub.3 A.sub.n : Oligomer of adenylic acid with 2',5'-phosphodiester linkages and a 5'-terminal triphosphate group.
A.sub.2, A.sub.3 and A.sub.4 : Dimer, trimer and tetramer of adenylic acid with 2',5'-phosphodiester linkages.
pA.sub.3, ppA.sub.3 (or p.sub.2 A.sub.3), pppA.sub.3 (or p.sub.3 A.sub.3): 5'-terminal mono-, di- and triphosphates of A.sub.3.
pA.sub.4, ppA.sub.4 (or p.sub.2 A.sub.4), pppA.sub.4 (or p.sub.3 A.sub.4): 5'-terminal mono-, di- and triphosphates of A.sub.4.
ApA: Dimer of adenylic acid with 2'-5'-phosphodiester linkage.
Ap*A: Dimer of adenylic acid with 2'-5'-phosphorothioate linkage.
PR: The R stereoconfiguration about a chiral phosphorous atom in a phosphorothioate internucleotide linkage.
PS: The S stereoconfiguration about a chiral phosphorous atom in a phosphorothioate internucleotide linkage.
A.sub.Rp *ApA: (PR)-P-thioadenylyl-(2',5')-adenylyl-(2',5')-adenosine.
A.sub.Sp *ApA: (PS)-P-thioadenylyl-(2',5')-adenylyl-(2',5')-adenosine.
ApA.sub.Rp *A: Adenylyl-(2',5')-(PR)-P-thioadenylyl-(2',5')-adenosine.
ApA.sub.Sp *A: Adenylyl-(2',5')-(PS)-P-thioadenylyl-(2',5')-adenosine.
pA.sub.Rp *ApA, ppA.sub.Rp *ApA, pppA.sub.Rp *ApA, pA.sub.Sp *ApA, ppA.sub.Sp *ApA, pppA.sub.Sp *ApA, pApA.sub.Rp *A, ppApA.sub.Rp *A, pppApA.sub.Rp *A, pApA.sub.Sp *A, ppApA.sub.Sp *A, pppApA.sub.Sp *A: 5'-mono-, di- and triphosphates of A.sub.Rp *ApA, A.sub.Sp *ApA, ApA.sub.Rp *A, and ApA.sub.Sp *A.
A.sub.Rp *ApApA: (PR)-P-thioadenylyl-(2',5')-adenylyl-(2',5')-adenylyl-(2',5')-adenosine.
A.sub.Sp *ApApA: (PS)-P-thioadenylyl-(2',5')-adenylyl-(2',5')-adenylyl-(2',5')-adenosine. Id.1 ApA.sub.Rp *ApA: Adenylyl-(2',5')-(PR) -P-thioadenylyl-(2',5')-adenylyl-(2',5')-adenosine.
ApA.sub.Sp *ApA: Adenylyl-(2',5')-(PS)-P-thioadenylyl-(2',5')-adenylyl-(2',5')-adenosine.
ApApA.sub.Rp *A: Adenylyl-(2',5')-(PR)-P-thioadenylyl-(2',5')-adenylyl-(2',5')-adenosine.
ApApA.sub.Sp *A: Adenylyl-(2',5')-adenylyl-(2',5')-(PS)-P-thioadenylyl-(2',5')-adenosine.
pA.sub.Rp *ApApA, ppA.sub.Rp *ApApA, pppA.sub.Rp *ApApA, pA.sub.Sp *ApApA, ppA.sub.Sp *ApApA, pppA.sub.Sp *ApApA, pApA.sub.Rp *ApA, ppApA.sub.Rp *ApA, pppApA.sub.Rp *ApA, pApA.sub.Sp *ApA, ppApA.sub.Sp *ApA, pppApA.sub.Sp *ApA, pApApA.sub.Rp *A, ppApApA.sub.Rp *A, pppApApA.sub.Rp *A, pApApA.sub.Sp *A, ppApApA.sub.Sp *A, pppApApA.sub.Sp *A: 5'-mono-, di- and triphosphates of the above tetramers.
bz: benzoyl PA1 ce: cyanoethyl PA1 CFS: Chronic Fatigue Syndrome PA1 DEAE: 2-(diethylamino)ethyl PA1 DBU: 1.8 diazabicyclo5.3.0!undec-7-enc PA1 HIV: Human Immunodeficiency Virus PA1 MeOTr: monomethoxytrityl PA1 M.O.I: multiplicity of infection PA1 mRNA: Messenger RNA PA1 npe: 2-(4-niytophenyl)ethyl PA1 PBL: Peripheral blood lymphocytes PA1 pCp: Cytidine 3'-5'-bisphosphate PA1 (PS)-ATP-alpha-S: Adenosine 5'O--(PS)-(1-thiotriphosphate). PA1 RNase L: 2-5A-dependent endoribonuclease. PA1 rRNA: Ribosomal RNA PA1 RT: Reverse transcriptase PA1 SCP: Specific cleavage products PA1 tbds: (tert-butyl)dimethylsilyl PA1 Tris: tris(hydroxymethyl)aminomethane PA1 tRNA: Transfer RNA
It is generally regarded that activation of RNase L by 2-5A is key to the antiviral defense mechanisms. Interferon induces transcription of the enzyme 2-5A synthetase which produces 2',5' linked oligoadenylates upon activation of double-stranded RNA. Previously, the only known biochemical effect of 2-5A is activation of RNase L. This enzyme hydrolyses mRNA and rRNA, thereby resulting in inhibition of protein synthesis. The activation of RNase L is transient unless 2-5A is continuously synthesized, since 2-5A is rapidly degraded. RNase L activation thus plays a critical role in inhibiting replication, and therefore in defending against infection by viruses.
A correlation has also been established between 2-5A metabolism and the growth cycle of HIV-1, i.e., high levels of 2-5A and activated RNase L correlate with failure of infected cells to release HIV-1, Schroder et al., J. Biol. Chem. 264: 5669-5673 (1989). Conversely, when the intracellular 2-5A pool decreases, RNase L can not be activated and HIV-1 production increases. A role for 2-5A cores as inhibitors of HIV-1 replication has been established with reports that 2-5A trimer and tetramer cores, 5'-monophosphates and 5'-triphosphates inhibit HIV-1 reverse transcriptase/primer complex formation, Montefiori et al., Proc. Natl. Acad. Sci. USA 86: 7191-7194 (1989); Muller et al., Biochemistry 30: 2027-2033 (1991); Sobol et al., Biochemistry 32: 1211-12118 (1993).
The introduction of the phosphorothioate group in the 2',5'-internucleotide linkages of 2-5A, induces metabolic stability greater than authentic 2-5A and resulted in the first 2-5A cores (i.e. 2-5A lacking 5'-phosphate moieties) able to activate RNase L (Kariko et al. Biochemistry 26: 7136-7142 (1987); Charachon et al. Biochemistry 29: 2550-2556 (1990)). Further, RNase L is a functionally stereoselective enzyme and 2-5A trimers and tetramers having at least one of the internucleotide phosphorothioate 2',5'-linkages of the PS configuration have greatly enhanced metabolic stability. The chemical synthesis of the fully resolved 2',5'-phosphorothioate adenylate trimer and tetramer cores has been reported, Suhadolnik et al., U.S. Pat. No. 4,924,624. Preparation of the stereoisomers via enzymatic synthesis is not possible due to the sterospecificity of 2-5A synthetase for the substrate (PS)-ATP-alpha-S, which yields trimer and tetramer products of the PR configuration exclusively. Further, while Lebleu et al., U.S. Pat. No. 4,981,957, discloses the enzymatic synthesis of a phosphorothioate-substituted derivative of 2',5' oligoadenylate, the compounds disclosed are not stereospecific.